ARF6 et ARF1 sont des petites GTPases de la famille des ARF(s) qui régulent plusieurs voies de signalisation comprenant, la formation et le mouvement des vésicules, la transformation des lipides membranaires et la réorganisation du cytosquelette d’actine. À ce jour, le rôle de la protéine ARF6 et de la protéine ARF1 dans la signalisation des récepteurs couplés aux protéines G (RCPG) et des récepteurs à activité tyrosine kinase (RTK) dans les cellules endothéliales est encore très peu étudié. Le but de cette étude a été de caractériser le rôle de la protéine ARF6 dans la migration des cellules endothéliales induite par l’endothéline-1, ainsi que le rôle de la protéine ARF1 dans la sécrétion du monoxyde d’azote (NO) stimulées par le VEGF. Dans cette étude, nous montrons qu’ARF6 est essentielle à la migration des cellules endothéliales induite par l’endotheline-1. L’inhibition de l’expression d’ARF6 par interférence à l’ARN entraîne une activation marquée de la kinase FAK et son association constitutive avec Src. Par ailleurs, cette inhibition affecte l’association entre GIT1 et la kinase FAK. Ceci se traduit par une inhibition du désassemblage des contacts focaux et une augmentation de l’adhésion cellulaire menant à une diminution de la motilité. De plus, nos résultats montrent que la protéine ARF1 est essentielle à l’activation d’eNOS et à la sécrétion du NO suite à l’activation du VEGFR2 dans les cellules endothéliales BAEC. En effet, l’inhibition de l’expression d’ARF1 par interférence à l’ARN entraîne une inhibition du recrutement de la kinase Akt à la membrane plasmique et une inhibition de son activation induite par le VEGF. L’inhibition de l’activation de la kinase Akt par le VEGF conduit à une inhibition de l’activation de eNOS et de la sécrétion du NO. Dans l’ensemble, nos résultats montrent que les protéines ARF6 et ARF1 sont essentielles à la signalisation de l’ETB et du VEGFR2 pour les processus menant à la migration cellulaire et à la sécrétion du NO respectivement, deux évènements essentiels à l’angiogenèse. / ARF6 and ARF1 are small GTPases of the ARF family(s) that regulate several signalling pathways including vesicles trafficking, lipid membrane remodelling and actin cytoskeleton reorganization. To date, the role of ARF6 and ARF1 in GPCR and RTK signalling, in endothelial cells, is little known. In this thesis, we aimed to characterize the role of ARF6 in the migration of endothelial cells induced by Enodothelin-1, and the role of ARF1 in the secretion of NO induced by VEGF. We show that ARF6 is essential for endothelial cell migration induced by endothelin-1. Inhibition of ARF6 expression using RNA interference markedly impaired basal and ET-1 stimulated cell migration. In this condition, FAK is found constitutively associated with Src. In contrast, depletion of ARF6 impairs the ability of GIT1 to form an agonist-promoted complex with FAK, thereby preventing disassembly of focal adhesions. As a consequence, adhesion of ARF6-depleted endothelial cells is increased and their motility is reduced. Furthermore, our result shows that ARF1 GTPase is essential for the activation of eNOS and the secretion of NO following VEGFR2 activation in endothelial cells. Inhibition of ARF1 expression using RNA interference markedly impaired the recruitment of Akt to the plasma membrane and its phosphorylation by the VEGF. As a consequence, the inhibition of Akt leads to an inhibition of eNOS, a well known downstream target, which in turn leads to inhibition of NO production. All together, our results indicate that ARF6 and ARF1 are essential for the ETB and the VEGFR2 signalling leading to cell migration and NO secretion respectively, two required steps for angiogenesis.

L'infarctus du myocarde constitue la première cause de mortalité cardiovasculaire. Ainsi, toute stratégie permettant de moduler la vulnérabilité du coeur à l'ischémie-reperfusion (IR) peut représenter un intérêt majeur de santé publique. L'exercice en endurance est reconnu comme une stratégie de cardioprotection efficace dont les mécanismes cellulaires restent néanmoins peu connus. Les objectifs de ce travail de thèse sont donc i) d'évaluer le rôle préventif de l'exercice sur le développement d'un phénotype sensible à l'IR myocardique, et ii) de tenter de mieux comprendre le rôle de la eNOS dans la radioprotection par l'exercice. Dans la première partie de ce travail, nous avons mis en évidence que l'exercice permet de prévenir le développement d'un phénotype pathologique cardiomyocytaire,par une amélioration du statut antioxydant et un maintien de l'homéostasie calcique cellulaire, et ainsi permet de normaliser la sensibilité du coeur à l'IR chez une population à risque. Dans un second temps, les travaux réalisés ont permis de mettre en avant le rôle majeur de la eNOS dans la cardioprotection par l'exercice. Cette cardioprotection est associée à une diminution du niveau de phosphorylation (Ser1177) et surtout de l'état de découplage de cette enzyme au cours des premières minutes de reperfusion. Ces modifications, associées à l'amélioration du statut antioxydant cardiaque par l'exercice, sont à l'origine d'une diminution du stress nitro-oxydant au cours de la reperfusion,expliquée par une moindre synthèse de NO et une meilleure capacité à éliminer l'O2.-, permettant ainsi de limiter la synthèse de ONOO-. L'ensemble de ce travail de thèse a ainsi permis de mettre en évidence la complexité de la cardioprotection par l'exercice, nécessitant l'interaction entre différents mécanismes cellulaires tels que l'amélioration du statut enzymatique antioxydant, le découplage de la eNOS au cours de la reperfusion précoce et la régulation de l'homéostasie calcique intracellulaire. Ce travail à d'autre part permis de mieux appréhender le rôle complexe de la voie de synthèse du NO parla eNOS dans la modulation de la vulnérabilité du coeur à un stress tel que l'IR

Submitted by Maria Medeiros (maria.dilva1@ufcg.edu.br) on 2018-08-13T15:28:50Z No. of bitstreams: 1 POLLYANNA KELLY DE OLIVEIRA SILVA - TESE (PPGMet) 2017.pdf: 11004478 bytes, checksum: 0d5e098181f432beffc2fd8155027f1e (MD5) / Made available in DSpace on 2018-08-13T15:28:50Z (GMT). No. of bitstreams: 1 POLLYANNA KELLY DE OLIVEIRA SILVA - TESE (PPGMet) 2017.pdf: 11004478 bytes, checksum: 0d5e098181f432beffc2fd8155027f1e (MD5) Previous issue date: 2017-08-30 / CNPq / O vento como fonte para geração de energia elétrica é analisado neste trabalho através de sua variabilidade e da obtenção de previsões de curto prazo para o ano de 2010, período de atuação de El Niño-Oscilação Sul (ENOS) moderado. Modelos de séries temporais propostos por Box-Jenkins e o indicador de desempenho de predição MMREE são usados para obter as melhores estimativas da velocidade do vento com base nas séries observadas. São utilizados dados anemométricos do Projeto SONDA situado às margens do Rio São Francisco em Petrolina – PE, e de dois parques eólicos localizados no litoral do Estado do Ceará: Quixaba (litoral leste), na cidade de Aracati, e Lagoa Seca (litoral oeste), na cidade de Acaraú. O ciclo diário do vento tem velocidades mais baixas (altas) no período da madrugada-início da manhã (pela manhã e final da noite, com exceção do litoral oeste, cujas máximas ocorrem no final da tarde). Um cisalhamento vertical negativo, no vento local, é observado em períodos distintos do dia nas três áreas de estudo. No Ceará ele ocorre no período da manhã (início da tarde e meio da noite) no litoral leste (oeste) e no Lago de Sobradinho durante a noite até o início da manhã. Foi observado que no litoral leste os ventos são mais fortes, provavelmente devido à curvatura côncava do litoral. As estimativas da velocidade do vento no horizonte de 24 horas pelo modelo SARIMA, com dados horários dos 30 dias anteriores ao dia da previsão para treino (Caso 2), mostraram redução nos erros e melhora significativa na série estimada no período da madrugada-início da manhã; no Lago de Sobradinho essas estimativas são mais precisas, quando comparadas àquelas feitas com base em toda a série de dados (Caso 1). Os resultados indicam que o modelo SARIMA com período de entrada de dados menor pode ser aplicado para a previsão da velocidade do vento em áreas de potencial eólico, dando suporte ao operador da rede elétrica na programação da geração despachável para o dia seguinte. / The wind as a source for power generation is analyzed in this work by means of its variability and short-range wind forecasts for the year of 2010, period of moderate El Niño-Southern Oscillation (ENSO). Time series models proposed by Box-Jenkins and the indicator of forecast accuracy MMREE are used to obtain the best wind speed estimates based on the observed series. Anemometric data of the SONDA Project located on the shore of the São Francisco River in Petrolina-PE, and of two wind power plants located on the coast of the Ceará State, Quixaba (east coast), in the city of Aracati, and Lagoa Seca (west coast), in the city of Acaraú, are used. The daily wind cycle has lower (higher) speeds in late night-early morning (in the morning and end of the night, with exception of the west coast, whose maxima occur in late afternoon). A negative vertical shear in the local wind is observed in distinct periods of the day in the three study areas. In Ceará it occurs in the morning (early afternoon and middle of the night) on the east (west) coast and on Sobradinho Lake at night until early in the morning. It was observed that the winds are stronger on the east coast, probably due to the coast’s concave curvature. The wind speed estimates in a 24-hour horizon by the SARIMA model, with hourly data of the 30 days that precede the forecast day for training (Case 2), showed reduction in the errors and significant improvement in the estimated series in late night-early morning; in Sobradinho Lake these estimates are more accurate, as compared to the estimates based on the entire data series (Case 1). The results indicate that the SARIMA model with horter time series as input may be applied to forecast wind speed in areas of eolic potential, giving support to the system operator in programming the dispatchable distributed generation for the next day.

Dentre as espécies florestais, o Eucalyptus é o gênero florestal mais plantado no Brasil, com aproximadamente 7,8 milhões de hectares, tendo grande importância econômica para o país. Desta maneira, é importante um melhor conhecimento e quantificação dos fatores que condicionam e reduzem o crescimento e a produtividade das florestas. Assim, este estudo teve por objetivos: i) adaptar, calibrar e avaliar o Modelo da Zona Agroecológica (MZA-FAO) para a estimação do crescimento e da produtividade de oito clones de eucalipto em diferentes regiões brasileiras; ii) determinar a magnitude e as principais causas das quebras de produtividade (yield gaps) da cultura do eucalipto em diferentes regiões produtoras do estado de Minas Gerais; e iii) avaliar a influência de eventos de El Niño, La Niña e Neutros na produtividade de eucaliptos em diferentes localidades produtoras do Brasil. Foram adaptados e calibrados os coeficientes do MZA-FAO, como a correção para o índice de colheita (Cc), o coeficiente de sensibilidade ao déficit hídrico (ky), as curvas características de índice de área foliar (IAF), crescimento radicular (Zr) e coeficiente de cultura (kc), além da inclusão de funções de penalização da produtividade do eucalipto por ocorrência de eventos de geada (ffrost) e mortalidade por longos períodos de deficiência hídrica (fwd). A inclusão das funções de penalização e a calibração dos coeficientes resultaram em uma melhora significativa no desempenho (acurácia e precisão) do modelo, com a REQM passando de 110 m3 ha-1, na fase inicial de calibração, para 39 m3 ha-1, na fase final de calibração, R2 passando de 0,73 para 0,82 e índice d indo de 0,70 para 0,93. Com relação às quebras de produtividade, a deficiência hídrica foi o principal fator de quebra de produtividade, correspondendo a 77% da quebra total (QT), enquanto que as perdas decorrentes do déficit de manejo corresponderam a 23% da QT. Para avaliar o crescimento do eucalipto em eventos de ENOS, no período de 1983 a 2016, foi considerado o Incremento Corrente Anual (ICA) no período de máximo crescimento, ou seja, no 3° ano de seu ciclo. As produtividades e as perdas de produtividade do eucalipto durante a atuação dos eventos de ENOS mostraram-se amplamente variáveis tanto espacialmente quanto temporalmente, como consequência dos diferentes regimes térmicos e hídricos das regiões produtoras, não sendo observado um padrão claro para a relação ENOS e produtividade do eucalipto em diferentes regiões brasileiras avaliadas. / Among the forest species, the Eucalyptus is the most planted one in Brazil, with approximately 7.8 million hectares, having a huge economic importance for the country. Therefore, a better knowledge and quantification of the factors that affect forests growth and yield is of high important. Based on that, this study had as objectives: i) to adapt, calibrate and evaluate the Agroecological Zone Model (AEZ-FAO) to estimate growth and yield of eight eucalyptus clones in different Brazilian producing regions; ii) to identify the magnitude and major causes of yield gaps of eucalyptus in different producing regions of Minas Gerais state, Brazil; and iii) to identify the influence of El Niño, La Niña and Neutral events on eucalyptus yield in 12 Brazilian producing regions. The model´s coefficients, such as correction for the harvest index (Cc), water deficit sensitivity index (ky), leaf area index curve (LAI), root growth curve (Zr) and crop coefficient (kc), were calibrated. Penalization functions for considering the occurrence of frost events (ffrost) and mortality by long periods of accumulated water deficit (fwd) were also included in the model. The calibration and adaptation of the model resulted in a significant improvement of its performance (accuracy and precision), for both calibration and evaluation phases. The RMSE was 110 m3 ha-1 in the initial phase of calibration while in the final phase, RMSE was reduced to 39 m3 ha-1, the R2 was increase from 0.73 to 0.82, and the agreement index (d) was also improved going from 0.70 to 0.93. In relation to the yield gaps, the water deficit was the main factor of yield reduction, corresponding to 77% of the total yield gap (YGT), while losses due to sub-optimum management corresponded to 23% of YGT. In order to evaluate the eucalyptus growth under different ENSO events, from 1983 to 2016, the Current Annual Increment (CAI) in the period of maximum growth, 3rd year of the cycle, was used. The eucalyptus yield and yield gap during the ENSO events were widely variable both spatially and temporally as a consequence of the different thermal and water regimes of the producing regions. It was not possible to identify a clear pattern thee relationship between ENSO and eucalyptus yield in the different Brazilian regions assessed.

A importância do Estado de São Paulo (ESP) é notável no desenvolvimento do Brasil, seja no setor econômico ou energético, o que justifica o estudo do comportamento do clima nessa região. O conhecimento da variabilidade da precipitação é imprescindível na gestão de recursos hídricos e possui grande impacto na agricultura e geração de energia por meio de fontes hidrelétricas. Estudos anteriores apontaram efeitos não-lineares do El Niño-Oscilação Sul (ENOS) sobre a precipitação no ESP; entretanto, nenhum estudo específico acerca da influência da Oscilação interdecadal do Pacífico (ODP) nesta área foi ainda realizado, embora haja alguns impactos conhecidos na América do Sul. Deste modo, este trabalho estudou a relação entre anomalias de precipitação no ESP e a ODP, no período de 1901 a 2007, de forma a auxiliar as pesquisas na linha da previsão climática nessa região do Brasil. Na primeira etapa, foram descritos os regimes de precipitação tanto para a América do Sul como localmente, para o ESP, onde se destacaram fatores como a topografia e a influência do Oceano Atlântico. Posteriormente, foram calculados quantis anuais e mensais que permitiram classificar cada evento quanto ao total de precipitação. Regiões pluviometricamente homogêneas foram determinadas no ESP com base na climatologia e nos quantis de precipitação. Notou-se a relação construtiva entre eventos ENOS e as fases da ODP, com máximo durante o verão austral. Os sinais da ODP são percebidos em todo o ESP principalmente na primavera e no verão austrais. Uma análise complementar mostrou que as fases da Oscilação Multidecadal do Atlântico (AMO) também contribuem para a precipitação no ESP durante o verão e a primavera austrais no litoral, durante o verão no interior, e ao longo da primavera na região da Serra da Mantiqueira. Aparentemente, não há relação entre os eventos ENOS e a AMO. / São Paulo State (SPS) is remarkably important to the development of Brazil, economically or energetically, and this justifies climate studies on that region. Knowing rainfall variability is essential to water resources management and it has a great impact on agriculture an power production by hydroelectric power plants. Previous studies have detected non-linear effects of El Niño-Southern Oscillation (ENSO) on SPS rainfall however no specific work deals with PDO influence in this area besides some impacts on South America are known. Therefore this work has studied the relationship between rainfall anomalies in SPS and PDO from 1901 to 2007 to contribute to the climate forecasting improvement. First it was described the rainfall regime in South America, and locally in SPS where topography and the Atlantic Ocean influences were of special importance. Then annual and monthly quantiles were calculated to allow the classification of events according to rainfall totals. Rainfall homogeneous regions were established in SPS using climatology and quantiles. It was observed the constructive relationship between ENSO events and PDO phases, mainly on austral summer. PDO signals were noticed all over the SPS mostly on austral spring and summer. An additional analysis showed that Atlantic Multidecadal Oscillation (AMO) phases also contribute to SPS rainfall during austral summer and spring at the coast, only on summer at the country and during spring at the Mantiqueira Slopes. Apparently, there is no relation between ENSO events and AMO phases.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A large-scale phenomenon that greatly affects the weather and the climate of different locations of the Globe and that has been widely studied during the last three decades is the El Niño Southern Oscilation (ENSO). ENSO leads to changes in the atmospheric circulation and affects climate variables, mainly rainfall, in all regions of Brazil. During El Niño years (the positive phase of ENSO), rainfall is often above normal in Southern Brazil whereas during La Niña years (the negative phase of ENSO), rainfall is often below normal. Some of the rainfall properties are affected by ENSO like frequency, intensity and amount. These properties are important to define if rainfall events are erosive. There are some methods of determining rainfall erosivity, among them the best and most used in Rio Grande do Sul State is the EI30 erosivity index. The objective of this dissertation was to determine and associate the EI30 erosivity index of rainfalls with the ENSO phenomenon in Santa Maria, RS, Brazil. Daily precipitation data recorded from 01 July 1978 to 30 July 2008 collected in a standard Climatological Station in Santa Maria, RS, were used. Individual erosive rainfall events were identified in the records, grouped into El Niño, La Niña and Neutral years, and had their EI30 calculated. Rainfalls were also grouped into three patterns: Advanced, Intermediate and Delayed. Pearson correlation and regression analyses between the EI30 erosivity index and the Niño Oceanic Index (NOI) were performed and the regression was tested with the t test in order to quantify the relationship between the two variables, aiming a possible predictability of the erosion potential of rainfall events from Sea Surface Temperature (SST) in the Pacific Ocean. Rainfall erosion potential in Santa Maria is affected by ENSO, so that a greater number of rainfall events have higher erosion potential during El Niño and neutral years. The variability of the rainfall erosion potential is greater during Neutral years than during years with SST anomalies. The frequency distribution of the EI30 erosivity index is skewed to the right in El Niño, La Niña and Neutral years. Rainfalls pattern is changed in years with SST anomalies, so that there is a slight increase in Advanced rainfalls during El Niño years and a slight decrease in Delayed rainfalls during La Niña years, whereas there is a decrease in rainfall events with Intermediate pattern during El Niño and La Niña years compared to Neutral years. The predictive capability of the erosion potential of rainfall events in Santa Maria from NOI index is weak or not possible. / Um dos fenômenos de grande escala que mais afetam o tempo e o clima de diferentes locais na superfície terrestre e que tem sido bastante estudado nas últimas três décadas é o fenômeno El Niño Oscilação Sul (ENOS). O ENOS provoca alterações na circulação atmosférica que afetam os elementos meteorológicos, principalmente a precipitação pluviométrica, em todas as regiões do Brasil. Em anos de El Niño, fase positiva do fenômeno, a precipitação pluviométrica é freqüentemente acima da normal na Região Sul do Brasil e em anos de La Niña, fase negativa do fenômeno, a precipitação pluviométrica é freqüentemente abaixo da normal. Algumas características das precipitações pluviométricas são afetadas pelo ENOS como a freqüência, a intensidade e a quantidade. Essas características são importantes para se definir se as chuvas são erosivas. Existem alguns métodos de determinação da erosividade das chuvas, através de índices de erosividade, entre os quais, o melhor e mais usado no Rio Grande do Sul (RS) é o Índice de Erosividade EI30. O objetivo desta dissertação foi determinar e associar o Índice de Erosividade EI30 das chuvas com o fenômeno ENOS para Santa Maria, RS. Usaram-se os dados de precipitação diária retirados de pluviogramas a partir de 01 de julho de 1978 a 30 de junho de 2008 coletados na Estação Climatológica Principal de Santa Maria, RS. As chuvas individuais e erosivas foram identificadas nos pluviogramas, classificadas em anos de El Nino, La Niña e Neutros e calculado o seu índice EI30. Também foram classificadas as chuvas em padrões de chuvas Avançado, Intermediário e Atrasado. Foi realizada a análise de correlação de Pearson e análise de regressão entre o Índice de Erosividade EI30 e o Índice Oceânico do Niño (ION) e a significância da regressão foi testada com o teste t com o objetivo de quantificar a associação entre as duas variáveis, com vistas à possível previsibilidade do potencial erosivo das chuvas a partir de anomalias de Temperatura da Superfície do Mar (TSM) no Oceano Pacífico. O potencial erosivo das chuvas em Santa Maria é afetado pelo fenômeno ENOS, de modo que um maior número de chuvas tem maior potencial erosivo em anos de El Niño e em anos Neutros. A variabilidade do potencial erosivo das chuvas em Santa Maria é maior nos anos Neutros do que nos anos de anomalia da TSM. A distribuição de freqüência do índice de erosividade EI30 de chuvas individuais é assimétrica positiva em anos de El Niño, La Niña e Neutros. O padrão das chuvas é alterado em anos de anomalia da TSM no sentido de que nos anos El Niño há um leve acréscimo nas chuvas de padrão avançado e em anos de La Niña há um leve acréscimo nas chuvas de padrão atrasado, e no padrão intermediário, decrescem o número de chuvas em anos de El Niño e La Niña comparado com anos Neutros. A capacidade preditiva do potencial erosivo das chuvas em Santa Maria pelo Índice ION é fraca ou inexistente.

No description available.

Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2009. / In the heart, endothelial nitric oxide synthase (eNOS) is regarded as the most important constitutively expressed enzymatic source of nitric oxide (NO), a major cardiac signalling molecule. On the whole, NO is regarded as a cardioprotective molecule. The role of eNOS during ischaemia / hypoxia is controversial; however, it is generally accepted that ischaemia / hypoxia results in increased cardiac NO production. Most studies focus either on the whole heart or isolated cell models. As yet, no study has compared findings with regard to NO metabolism in these two distinct models, in a single study. We hypothesise that observations in a whole heart model with regard to increased NO production and eNOS involvement in ischaemia are the result of events on cellular level and that the increase in NO production observed during hypoxia in cardiomyocytes and endothelial cells is at least in part due to the increase in expression and / or activation of eNOS. Furthermore, we hypothesize that these effects are mediated via the PI3-K / PKB pathway. We aimed to measure and compare NO-production and eNOS expression and activation in the whole heart and isolated cardiac cells and measure PKB expression and activation in the cells under normoxic and ischaemic / hypoxic conditions. We also aimed to determine the effects of PI3-K / PKB pathway inhibition on NO production and eNOS expression and activation in isolated cardiac cells under normoxic and hypoxic conditions. Adult rat hearts were perfused and global ischaemia induced for 15 and 20 min. Tissue homogenates of perfused hearts were used for the measurement of nitrites and determination of expression and activation of eNOS. Expression of eNOS in the heart was also determined by immunohistochemical (IHC) analysis. Cardiomyocytes were isolated from adult rat hearts by collagenase-perfusion, and adult rat cardiac microvascular endothelial cells (CMEC) purchased commercially. In the cells, hypoxia was induced by covering cell pellets with mineral oil for 60 min. Cell viability was determined by trypan blue and propidium iodide (PI) staining and intracellular NO production measured by FACS analysis of the NO-specific probe, DAF-2/DA and by measurement of nitrite levels (Griess reagent). Results show that in ischaemic hearts, nitrite production increased by 12 % after 15 min ischaemia and 7 % after 20 min ischaemia. Total eNOS expression remained unchanged (Western Blot and IHC) and activated eNOS (phospho-eNOS Ser1177) increased by 38 % after 15 min ischaemia and decreased by 43% after 20 min ischaemia. In the cells, both viability techniques verified that the hypoxia-protocol induced significant damage. In isolated cardiomyocytes, NO-production increased 1.2-fold (by DAF-2/DA fluorescence), total eNOS expression increased 2-fold and activated eNOS increased 1.8-fold over control. In CMECs, NO-production increased 1.6-fold (by DAF-2/DA fluorescence), total eNOS increased by 1.8- fold and activated eNOS by 3-fold. With regards to our PI3-K / PKB investigations, results showed an increase of 84 % and 88 % in expression vii and activation of PKB (phospho Ser473) in hypoxic cardiomyocytes, respectively. In hypoxic CMECs, there was no change in PKB expression but there was a 69 % increase in phosphorylated PKB. NO production in wortmannin-treated hypoxic cardiomyocytes decreased by 12 % as compared to untreated hypoxic cells. In treated hypoxic CMECs, NO production decreased by 58 % as compared to untreated hypoxic cells. Treatment with wortmannin did not change the expression of eNOS protein in the cardiomyocytes, however, activated eNOS decreased by 41 % and 23 % under baseline and hypoxic conditions in treated cells respectively. There was a significant increase in NO production after exposure to O2 deficient conditions in all models investigated, a trend similar to what previous studies in literature found. However, the source of this NO is not fully understood although it has been discovered that NOS plays a role. Our data reveals similar trends in 15 min ischaemia in whole hearts and 60 min hypoxia in the cells; however, the trends observed at 20 min ischaemia are in conflict with our cell data (i.e. decrease in activated eNOS). This may be due to the severity of the ischaemic insult in whole hearts and/or the presence of other cell types and paracrine factors in the whole heart. Hypoxia increased the activation of PKB in the isolated cardiac cells. Inhibition of the PI3-K / PKB pathway reduced NO production and hypoxia-induced eNOS activation in cardiomyocytes. In conclusion, we have, for the first time, demonstrated that the increase in NO production during hypoxia is due (at least in part) to an increase in eNOS phosphorylation at Ser1177 and that this is mediated via the PI3-K / PKB pathway.

O evento climático conhecido como El Niño Oscilação Sul (ENOS) é formado pelos episódios de El Niño e La Niña e é classificado como um fenômeno de grande escala que ocorre no Oceano Pacífico Equatorial. Em razão do grande efeito do fenômeno ENOS na variabilidade climática e, consequentemente, na produção agrícola, se faz necessário o conhecimento adequado das consequências dos eventos de El Niño e La Niña nos regimes térmicos e hídricos de diferentes regiões brasileiras e de seus impactos na produção de alimentos, sobretudo na cultura da cana-de-açúcar. Para tanto, dados meteorológicos foram analisados a fim de se verificar algum efeito causado pelos eventos do ENOS na temperatura do ar, na radiação solar e precipitação pluvial. Em seguida, utilizou-se o modelo DSSAT CSMCANEGRO parametrizado para as condições brasileiras para simular a produtividade da cana-planta de 12 meses em quatro localidades de diferentes regiões do Brasil (Jataí, GO; João Pessoa, PB; Londrina, PR; e Piracicaba, SP), empregando-se séries históricas de dados meteorológicos, de 1979 a 2010, para três tipos de solos com diferentes características físico-hídricas (capacidade de água disponível), e para dois tipos de simulação da produtividade da cana pelo modelo DSSAT CSM-CANEGRO, o tipo Seasonal e o tipo Sequence. Foi possível notar nos resultados que para a temperatura do ar houve uma maior frequência de anos com essa variável acima da mediana nas localidades situadas na região central e nordeste no país durante os eventos de El Niño, ao passo que na região sul, representada por Londrina, tal frequência foi indefinida. Para os anos de La Niña, não houve, em geral, tendência clara de variação em nenhuma das localidades. Já nos anos neutros as maiores frequências foram de temperaturas abaixo da mediana nas localidades das regiões central e sul, enquanto em João Pessoa, PB, não houve tendência bem definida. Para a radiação solar, em geral, não se detectaram tendências expressivas, apesar de valores levemente acima da mediana em anos de La Niña, em todas as regiões. Finalmente, para as chuvas houve tendências um pouco mais expressivas, sendo que nas localidades da região central do país (Jataí e Piracicaba) as precipitações acima da mediana foram mais frequentes nos anos de El Niño e La Niña, ficando abaixo da mediana nos anos neutros. Nas demais localidades analisadas, as chuvas tenderam a ficar abaixo ou igual à mediana durante todas as fases do ENOS. Quanto à produtividade, algumas tendências também puderam ser observadas. Em Jataí, GO, não houve alterações da produtividade média maiores do que ± 1 t ha-1. Em João Pessoa, PB, a tendência de menores produtividades durante os anos de El Niño e de La Niña e de maiores produtividades em anos neutros. Situação oposta foi observa em Piracicaba, SP, e Londrina, PR, onde as produtividades tenderam a serem maiores do que a média histórica nos eventos tanto de El Niño como de La Niña, ao passo que nos anos de neutralidade do ENOS as produtividades tenderam a ser menores do que a média. / The climatic event known as El Niño Southern Oscillation (ENSO) is formed by episodes of El Niño and La Niña and is classified as a large-scale phenomenon that occurs in the Equatorial Pacific Ocean. Given the large effect of ENSO on climate variability and hence in agricultural production, proper knowledge of the consequences of El Niño and La Niña on the thermal and water regimes of different Brazilian regions and their impact on food production is needed, especially for sugarcane crop. To this end, climate variables were analyzed in order to verify any effect caused by the ENSO events on air temperature, solar radiation and rainfall. The DSSAT CSM-CANEGRO model, parameterized for the Brazilian conditions, was used to simulate sugarcane yield (plant cane of 12 months) in four sites of different Brazilian regions (Jataí, GO; João Pessoa, PB; Londrina, PR; and Piracicaba, SP), using historical series of meteorological data from 1979 to 2010, for three types of soils, with different physical-hydric properties (Soil water holding capacity), and two types of simulations in DSSAT CSM-CANEGRO model, with the Seasonal and Sequence procedures It was possible to notice in the results that the air temperature was a greater frequency of years with this variable above the median in localities situated in the central and northeastern region of the country during the El Niño events, while in the south, represented by Londrina, this frequency was undefined. For La Niña years, there was generally clear trend of variation in any of the locations. Already in neutral years, the highest frequency was below the median temperatures in the localities in central and southern regions, while in João Pessoa, PB, and no well-defined trend. Solar radiation, in general, no significant trends were detected, although values slightly above the median in La Niña years in all regions. Finally, to the rains there was a little more expressive tendency, and the locations of the central region (Jataí and Piracicaba) precipitation above the median were more frequent in years of El Niño and La Niña, below the median in neutral years. In other areas analyzed, rainfall tended to be below or equal to the median during all phases of ENSO. With regard to productivity, some trends were also observed. In Jataí, GO, no changes greater than ± 1 t ha-1 was observed. João Pessoa, PB, there was a trend of lower yields during El Niño and La Niña years and higher yields during neutral years. Opposite situation was observed in Piracicaba, SP, and Londrina, PR, where the yields tended to be higher than the historical average in both El Niño and La Niña events, while during neutral years the yield tended to be smaller than average.

Store-operated calcium entry (SOCE) is a principal mechanism for extracellular calcium entry in non-excitable cell types, and is primarily facilitated by the calcium- release activated calcium (CRAC) channel; itself comprised of the pore-forming Orai-1 and calcium-sensing Stromal interaction molecule (STIM)-1 proteins. Depletion of endoplasmic reticulum (ER) calcium stores initiates STIM-1 translocation to defined ER-plasma membrane puncta, and subsequent Orai-STIM interaction and opening of Orai. The importance of this mechanism in calcium signalling in diverse tissue types is becoming increasingly clear. The vascular endothelium is a dynamic tissue, involved in the maintenance of vascular homeostasis and haemostasis. Many endothelium-derived bioactive agents, such as endothelin-1, prostaglandins, and the potent vasodilator nitric oxide (NO), are known to be produced via calcium- dependent mechanisms. However, the role of the CRAC channel in the vascular endothelium is poorly defined with little known about downstream targets of calcium influx through CRAC channels. The dysregulation of NO production by endothelial nitric oxide synthase (eNOS) is a major contributory factor in many vascular disease states, yet the calcium channel responsible for eNOS activation has yet to be identified. Within this thesis, I establish the endothelial cell line sEnd.1 as a new model system for studying CRAC channel signalling in the vascular endothelium, defining sEnd.1 SOCE as being CRAC channel-dependent. Inhibition of CRAC channels with an array of inhibitors, and knock-down of STIM-1, both reduced ATP- and TG-induced SOCE. The sEnd.1 model system was subsequently used to identify calcium entry through the CRAC channel as the elusive activation mechanism for eNOS. Through real-time imaging with the fluorescent NO dye DAF-2-DA, we established that NO production is non-linear, with a slow initial increase preceding a faster NO production phase. These kinetics, with a characteristic delay before fast production have, to our knowledge, not previously been reported. The time taken to reach the fast phase of NO production could be manipulated through changes in both local and bulk calcium rises, which indicated roles for both elements of calcium signalling in eNOS activation. eNOS regulation by calcium is complex, occurring not only through direct binding of calcium-calmodulin, but additionally through changing post-translational modifications, which in turn regulate the calcium-dependency of eNOS, such as phosphorylation of Ser1177. We propose that the delay in fast production of NO is due to the time taken to alter eNOS post-translational modifications, which thus remove inhibition on eNOS. Activation of CRAC channels increased phosphorylation of residue Ser1177 via calcium-calmodulin kinase II (CaMKII), with a similar time course to that required to reach the fast phase of NO production. Inhibition of CaMKII increased the time taken to reach fast activation. In conclusion this thesis presents a new model system for investigation of CRAC channel signalling in the endothelium. Furthermore, we clearly identify a critical endothelial pathway as being regulated by CRAC channels, by demonstrating the production of NO in response to both ATP and TG, which stimulate calcium entry through CRAC channels.